Composition and method for determination of albumin in fluids, particularly urine



United States Patent CCMPCSETIGN AND METHCD FCR D t'iERMlNA- TlGN CF ALBUM- EN EN FLUEDS, PARTICULARLY URINE Richard S. Niels-oils, Dale E. Farmer, and Paul W. Gerding, Elldiart, Ind., assignors to Miles Laboratories, Inc., Elkhart, Ind, a corporation of lndiana No Drawing. Filed Apr. 12, 1956, Ser. No. 577,671

3 Claims. (Cl. 2.3--230) This invention relates to a composition and to a method of using that composition for the determination of albumin in body fluids such as urine, for example.

Inasmuch as the presence of albumin in urine is an indication of a diseased or traumatic condition, the utility of a simple test for the determination of albumin is ob- The methods of present day use for the determination of albumin in urine consist of the following qualitative tests:

(a) Heat test with acetic acid which involves boiling a measured quantity of urine and then adding equal amounts of acetic acida turbidity is formed in a positive specimen.

Hellers test which involves placing an amount of concentrated nitric acid in a test tube and stratifying a small amount of urine on the nitric acid. A cloudy ring at the junction of the two liquids indicates a positive specimen.

(0) Sulfosalicylic acid test which requires the placing of 4 or 5 ml. of urine in a test tube and adding two or three drops of an aqueous solution of sulfosalicylic acid and a turbidity indicating a positive specimen.

(d) Picric acid test which involves placing a measured amount of urine in a test tube and adding an equal amount of saturated picric acid solution. Turbidity of the solution indicates a positive specimen.

The foregoing, and modifications thereof, constitute the presently employed methods of determination of the presence of albumin in urine, all of which depend on a turbidity test. In these, as indicated above, the albumin is precipitated either as a ring at the junction of the specimen and the precipitating agent, or as a suspension wherein the specimen is thoroughly mixed with the precipitating reagent.

Now, in order to circumvent the diiliculties inherently involved in turbidity testing, to eliminate the preparation of solutions, and to avoid handling of corrosive acids, we have developed a composition and method for albumin determinations that is unique in its ease of application. The nature of the composition and method of use is such that an unskilled person can conduct the test and can, moreover, readily run mass screening test determination on large groups of urine specimens. These and related advantages are achieved by the present invention by means of a color test, a color test which is purely of the changing color variety rather than of the turbidity type.

In particular, the objects and advantages sought are obtained by the use of a tabletted composition which indicates concentrations of albumin in urine when only one or two drops of the specimen to be tested is placed upon the tablet. The composition of that tablet in general includes a protein precipitant comprising a member of one or more of the following groups: (1) a strong, solid, water-soluble acid such as suliosalicylic acid or picric acid, (2) the alkali metal phosphates as are particularly represented by sodium tetrametaphosphate or sodium hexametaphosphate, and (3) metal salts such as lead nitrate, zinc sulfate, barium chloride, aluminum sulfate or aluminum chloride. A second ingredient of the present composition comprises, an eliervescent couple consisting of a strong, solid, water-soluble acid such as those listed above as protein prccipitants or maleic, tartaric,

citric, itaconic, sulfamic and like acids, together with an alkali metal carbonate or bicarbonate, the effervescent couple being eflective to produce carbon dioxide in an aqueous medium. The third primary ingredient is a dye component consisting of one or a plurality of dyes at least one of which has an atlinity for the protein as precipitated from solution to the extent that it is adsorbed upon the precipitated protein. In addition to its afiinity for the protein being precipitated, one or more of the dyes constituting the dye component must exhibit a color change from its acid to its basic color within the range of pH increase which the precipitated albumin uniquely exhibits.

Additional description of the concepts and conditions for practicing the invention will follow the ensuing detailed description of representative formulae in parts by weight of the compositions contemplated by this invention.

Examples Monosodium salt of i-m-sulloplienylazo-diphenylamine.

VII VIII Citric acid, anhyd Sulfosslieylic acid, anhyd Calcium sulfate Boric acid Sodium bicarbona c Sodium tetrametaphosphate Sullamie aeid Brilliant Green F D C Violet #1 .03 p-(p-Anilinophenylazo) benzene sulfonic acid sodium salt Crystal Violet Possibly the preferred formula, but which at the same time is subject to variations, is the following in conjunction with which we have indicated the degrees of variation permissible in the several ingredients even to the point of eliminating certain of the constituents entirely:

Example IX In all of the foregoing examples, precipitation or" the protein was effected below its iso-electrie point. in particular, sulfosalicylic acid, picric acid, sodium tetrametaphosphate and sodium hexametaphosphate are examples of chemicals which will precipitate protein below the isoeleetric point, the sodium tetrametaphosphate and hexametaphosphate requiring additional acidity such as is provided by sulfamic or citric acids, for example. When precipitating the albumin below its iso-electric point, the reactant mixture should be at a pH no higher than 3 and consequently the dye component must change from the acidic color to the basic color at or below a pH of 3. In practice, the optimum pH for the precipitation appears to be in the range of 2.0 to 2.5.

7 parts by weight:

triphenylainine) and Pi'opyl Red (anthranilic acid-di hpropyl aniline) separately as the indicator dye although dye component. At least one of the dyes, "where more here again it is a contemplated possibility that any of than one dye is employed, must have an afilnity for prothese representative dyes may be used in combination tein and must also have sufiicient tinctorial strength to 5 with other dyes to form a dye component. In general, a make color changes sufficiently definite for easy, accurate dye which changes color bo a pH of 6 ill not give readings. Dyes which possess these desirable or essena color difference inasmuch as a pH of 6 seems to be the tia-l attributes are Brilliant Green (sulfate salt of tetrahighest pH at which the method and composition will ethyldiaminotriphenylmethane), Crystal Violet (hexa- Work when the protein is precipitated above its iso-elecmethylpararosaniline chloride), Cresol Red (o-cresolsu ltric point.

fonphtha-lein), p-(p-anilinophenylazo) benzene sulfonic To again illustrate the variations that are possible in acid-sodium salt, Malachite Green (zinc double oxalate vformulae where precipitation of the protein occurs above of tetramethyl-p-aminotriphenyl-carbinol, m-(p-anilinothe iso-electric point, we have selected a preferred torphenylazo) benzene sulfonic acid-sodium salt, Brilliant As indicated in the earlier general discussion, not all dyes changing color below a pH of 3 can be used in the mula and have included possible variations of each in- B-lue (disodium salt of 4-([4-N-ethyl-p-sulfobenzylamil gredient in parts by weight, as follows:

no) phenyl] (2 sulfoniumphenyl) methylene)-[l- (N ethyl N p sulfobenzyl) 1 cyclohexadienimine], FD & C Violet #1, and Tartrazine (trisodi-um salt Example XVIII of 3-carboXy-5-hydroXy-l-p-sultophenyl-4-p-sulfopheny1- Variation azopyrazole). Metanil Yellow '(monosodium salt of 4-m- I sulfophenylazodiphenylaniine) and Basic Fuchsine (a Citric acid 5 3-7 mixture of rosaniline and pararosaniline hydrochlorides) l-fig are examples of dyes that are suitably adsorbed upon the Sodium bicarbonate. 3 2-5 Sodium citrate 14 11-18 precipitated protein but which give a more difficultly determinable result because of their weak tinctorial effect.

In Example IX, the sulfosalicylic acid and the sodium hexametaphosphate, both separately and together, precipitate the protein, the acid providing sufilcient acidity for Q lhhthasthig P i Point hlghhl' thal} that of t the hexametaphosphate to function. As an economical l t h tahlet- In either Case, the PlathCe Q the hmeasure, the cheaper citric acid is included to provide 15 the Same; one or two drops 0f the urine 1 additional acidity both for the pH regulation necessary men are the tahltt- Functionally W f protein precipitation by the hexametaphosphate and the sodium bicarbonate which with the acidspresent for reaction with the sodium bicarbonate to produce forms an effervescent couple, Provides a means for the effervescence. In this particular formula, Tartrazine is T816935? of the active ihgredthhts of the tablets h E1150 included only to dif the background color f the provides a controlled mechanical means for the solation tablet whereas Brilliant Green provides the color change of the lhtciPith-ted Protein Whlch W111 P055655 distinctive color in the case of a positive specimen. Through efferfor the reading of the test. Calcium sulfate is used as a n and bm-ic acid as a lubricant to aid tabletting' vescence at the tablet surface, the precipitated protein For precipitating albumin above its iso-electric point, 40 cohl-ehttated and isolated on p of the l P- we have found that lead nitrate, Zinc sulfate, barium chlo- Patehtly, tit the momhht of its P P the albumin ride, aluminum sulfate and aluminum chloride exemp ify P Suthvieht Carbon thOXtdc to cause the albumin the water-sol-ublesalts of metals that willfunction in'this to fish to the Surface and remain conversely in role. Specific formulae which are representative of Case of a hegativhspecimeh, foaming shhsidhs more other operable compositions include the following in quickly, and Within a Seconds Shecihcahy the liquid remaining on the tablet being free of bubbles and being, of course, of the same color as the original solution. The latter effect as it applies to the liquid itself is always the case in .both negative and positive specimens. For example, in the case of the formula containing Brilliant Green as the dye component, the foam which rises from the tablet surface will become blue, the amount and intensity depending on the quantity of albumin present. The tablet and liquid underneath the foam will remain It can thus be seen that the precipitation of albumin can be accomplished either below or above its iso-electric point with no resulting difference in the proteins property Examples Citric acid Sodium acetate .i Methyl red (p-dinethylarnino-azobenzeneo'ea-rboxylic acid) Sodium bicarbonate ii iiiiii i ifijil an orange-yellow and the foam, if present, will also be gromercsolGreenfl orange-yellow in a negative specimen. With Crystal f g fgg gg Violet, the color change is from the original purple to a Aluminum sulfate. green positive; for Basic Fuchsine the color change is Sodium wrath from pale yellow to a light red; and for External D & C

Yellow No. 1 the color changes from violet to yellow. For any dye that is selected, the change will be from its acid color to its basic color in positive specimens within the capacity and range of pH increase effected by the ggg g g -g g g g g precipitated protein as has been earlier discussed.

Sodium u fi 9 Although we have described our invention in relation sulfat g g g g to urinalysis, it is evident from the broad principles taught a g g sjijj 6 7 11 that modifications can be readily made Where necessary 4 -(4 -dhnethylamino -1 -nap oxy-benzene sulionic acid. Resazurin D ia zoresoreinol Lacinoid CsHz(OH) s-N-[CnHstOIU 212. .e Propyl Red (anthranilic acid di-n-propyl aniline). T

to extend the area of application of the method and composition to other body fluids. Within the area of this and other obvious modifications of the invention we claim:

1. A method for the determination of albumin in The foregoing are illustrative examples using Bromcresol Green (tetrabromo m cresolsulfon -phthalein), liquid on a tableted mixture of a protein precipitant, a Resazurin (Diazoresorcinol), Lacmoid (heptahydroxy- 7 dye having an affinity for the precipitated protein and a liquid which comprises depositing a test portion of the which exhibits a color-change Within the variation of pH effected by the precipitated protein, and an effervescent couple effective When moistened by said liquid to release carbon dioxide, said carbon dioxide forming with said liquid a foam on the surface of said tablet, said foam including the dyed albumin precipitated from said liquid.

2. In a method for determining albumin in a liquid, the step which comprises precipitating albumin from said liquid by depositing a test portion of the liquid on a tablet comprising a mixture of a protein precipitant, a dye having an aflinity for the precipitated protein and which exhibits a color change Within the variation of pH effected by the precipitated protein, and an effervescent couple eifective when moistened by said liquid to release carbon dioxide, whereby said carbon dioxide forms a foam with said liquid on the surface of said tablet, said foam including dyed precipitated albumin.

3. A composition for determining the presence of albumin in liquids, comprising a tableted mixture of a protein precipitant selected from the group of metal salts consisting of lead nitrate, zinc sulfate, barium chloride, aluminum sulfate and aluminum chloride, a dye having an afiinity for the precipitated protein, said dye exhib-it ing a color change within the range of pH increase effected by such precipitated protein, and an eflervescent couple consistin essentially of a strong Water-soluble onganic acid and a member of the group consisting of alkali metal carbonates and bicarbonates.

References Cited in the file of this patent UNITED STATES PATENTS (1951), Chas. C. Thomas Publ, 301327 E. Lawrence Ave, Springfield, Illinois, pp. 461465.

Kolmer: Approved Lab. Tech, 1951, 5th ed., p. 142. Free: Gastroenterology, vol. 24, pp. 414 to 421, July Peigl: Chem. of Specif. Sel. and Sensitive Reactions, 1949, p. 485.

Van Gool: Chem. Weekbl, 1950, pp. 7047.

Bohle: Klin. Wochschrift, 1953, vol. 31, pp. 798802.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No 3, 121 g 612 February 18 1964 Richard So Nicholls et a1 It is hereby certified that error appears in the above numbered patent requiring correction and that the said Letters Patent should read as corrected below Column 1 line 70 for "composition" read compositions column 2 in the table, under the headings "I" "III" III" an "IV" for "8", each occurrence, read 08 o Signed and sealed this 1st day of September 1964,

(SEAL) Attest:

ERNEST W. SWIDER EDWARD J. BRENNER Attosting Officer Commissioner of Patents 

1. A METHOD FOR THE DETERMINATION OF ALBUMIN IN A LIQUID WHICH COMPRISES DEPOSITING A TEST PORTION OF THE LIQUID ON A TABLETED MIXTURE OF PROTEIN PRECIPITANT, A DYE HAVING AN AFFINITY FOR THE PROTEIN AND WHICH EXHIBITS A COLOR-CHANGE WITHIN THE VARIATION OF PH EFFECTED BY THE PRECIPITATED PROTEIN, AND AN EFFERVESCENT COUPLE EFFECTIVE WHEN MOISTENED BY SAID LIQUID TO RELEASE CARBON DIOXIDE, SAID CARBON DIOXIDE FORMING WITH SAID LIQUID A FOAM ON THE SURFACE OF SAID TABLET, SAID FOAM INCLUDING THE DYED ALBUMIN PRECIPITATED FROM SAID LIQUID. 